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Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants

Tyrosinase-related protein 2 (Tyrp2) is involved in the melanogenesis pathway, catalyzing the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Recently, a new type of albinism was discovered with disease-causing mutations in the TYRP2 gene. Here, for the first time, we...

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Autores principales: Dolinska, Monika B., Woods, Taariq, Osuna, Isabella, Sergeev, Yuri V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8836267/
https://www.ncbi.nlm.nih.gov/pubmed/35163231
http://dx.doi.org/10.3390/ijms23031305
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author Dolinska, Monika B.
Woods, Taariq
Osuna, Isabella
Sergeev, Yuri V.
author_facet Dolinska, Monika B.
Woods, Taariq
Osuna, Isabella
Sergeev, Yuri V.
author_sort Dolinska, Monika B.
collection PubMed
description Tyrosinase-related protein 2 (Tyrp2) is involved in the melanogenesis pathway, catalyzing the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Recently, a new type of albinism was discovered with disease-causing mutations in the TYRP2 gene. Here, for the first time, we characterized the intra-melanosomal protein domain of Tyrp2 (residues 1-474) and missense variants C40S and C61W, which mimic the alterations found in genetic studies. Recombinant proteins were produced in the Trichoplusia Ni (Ti. Ni) larvae, purified by a combination of immobilized metal affinity (IMAC) and gel-filtration (GF) chromatography, and biochemically characterized. The mutants showed the protein expression in the lysates such as the wild type; however, undetectable protein yield after two steps of purification exhibited their misfolding and instability. In addition, the misfolding effect of the mutations was confirmed computationally using homology modeling and molecular docking. Together, experiments in vitro and computer simulations indicated the critical role of the Cys-rich domain in the Tyrp2 protein stability. The results are consistent with molecular modeling, global computational mutagenesis, and clinical data, proving the significance of genetic alterations in cysteine residues, which could cause oculocutaneous albinism type 8.
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spelling pubmed-88362672022-02-12 Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants Dolinska, Monika B. Woods, Taariq Osuna, Isabella Sergeev, Yuri V. Int J Mol Sci Article Tyrosinase-related protein 2 (Tyrp2) is involved in the melanogenesis pathway, catalyzing the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Recently, a new type of albinism was discovered with disease-causing mutations in the TYRP2 gene. Here, for the first time, we characterized the intra-melanosomal protein domain of Tyrp2 (residues 1-474) and missense variants C40S and C61W, which mimic the alterations found in genetic studies. Recombinant proteins were produced in the Trichoplusia Ni (Ti. Ni) larvae, purified by a combination of immobilized metal affinity (IMAC) and gel-filtration (GF) chromatography, and biochemically characterized. The mutants showed the protein expression in the lysates such as the wild type; however, undetectable protein yield after two steps of purification exhibited their misfolding and instability. In addition, the misfolding effect of the mutations was confirmed computationally using homology modeling and molecular docking. Together, experiments in vitro and computer simulations indicated the critical role of the Cys-rich domain in the Tyrp2 protein stability. The results are consistent with molecular modeling, global computational mutagenesis, and clinical data, proving the significance of genetic alterations in cysteine residues, which could cause oculocutaneous albinism type 8. MDPI 2022-01-24 /pmc/articles/PMC8836267/ /pubmed/35163231 http://dx.doi.org/10.3390/ijms23031305 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Dolinska, Monika B.
Woods, Taariq
Osuna, Isabella
Sergeev, Yuri V.
Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants
title Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants
title_full Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants
title_fullStr Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants
title_full_unstemmed Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants
title_short Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants
title_sort protein biochemistry and molecular modeling of the intra-melanosomal domain of human recombinant tyrp2 protein and oca8-related mutant variants
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8836267/
https://www.ncbi.nlm.nih.gov/pubmed/35163231
http://dx.doi.org/10.3390/ijms23031305
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